Off-the-shelf third-party HSC-engineered iNKT cells for ameliorating GvHD while preserving GvL effect in the treatment of blood cancers

Summary Allo-HSCT is a curative therapy for hematologic malignancies owing to GvL effect mediated by alloreactive T cells; however, the same T cells also mediate GvHD, a severe side effect limiting the widespread application of allo-HSCT in clinics. Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity. Here, we report the successful generation of third-party HSC-engineered human iNKT (3rdHSC-iNKT) cells using a method combining HSC gene engineering and in vitro HSC differentiation. The 3rdHSC-iNKT cells closely resembled the CD4−CD8−/+ subsets of endogenous human iNKT cells in phenotype and functionality. These cells displayed potent anti-GvHD functions by eliminating antigen-presenting myeloid cells in vitro and in xenograft models without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia, supporting 3rdHSC-iNKT cells as a promising off-the-shelf cell therapy candidate for GvHD prophylaxis.

generated off-the-shelf at high yield and purity 3rd HSC-iNKT cells ameliorate GvHD while preserving GvL effect 3rd HSC-iNKT cells effectively target antigenpresenting myeloid cells through CD1d 3rd HSC-iNKT cell product is a therapy candidate for GvHD prophylaxis in allo-HSCT

INTRODUCTION
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematologic malignancies such as leukemia/lymphoma owing to the graft-versus leukemia/lymphoma (GvL) effect elicited by alloreactive donor T cells (Appelbaum, 2001;Gribben and O'Brien, 2011;Shlomchik, 2007). In 2018 alone, more than 47,000 bone marrow transplantations were performed worldwide, 19,000 (41%) of which were allogeneic and nearly all for the treatment of leukemia/lymphoma (Passweg et al., 2020). However, the development of graft-versus-host disease (GvHD) mediated by alloreactive donor T cells responding to minor or major histocompatibility antigen disparities between donor and recipient remains a major cause of patient morbidity and mortality for patients receiving T-cell replete allo-HSCT (Chakraverty and Sykes, 2007;Ferrara et al., 2009;Hill et al., 2021). T cell depletion of the graft can reduce the incidence and severity of GvHD in patients but is associated with an increased risk of graft rejection, infections, and leukemia relapse (Apperley et al., 1986). Therefore, extensive research has been focused on identifying other cellular components of the graft that could modulate donor T cells and reduce the risk and severity of GvHD without diminishing normal immunological functions, including NK (Yamasaki et al., 2003), B (Shimabukuro-Vornhagen et al., 2009), and CD4 + CD25 hi FoxP3 + T regulatory (Treg) cells (Pabst et al., 2007;Wolf et al., 2007).
Invariant nature killer T (iNKT) cells have also been studied extensively for their roles in modulating GvHD and GvL. iNKT cells are a small subset of ab T cells that express both a semi-invariant T cell receptor (TCR) (Va24-Ja18 in humans and Va14-Ja18 in mice paired with a limited selection of Vb chains) and natural killer cell markers (e.g., CD161 in humans and NK1.1 in mice) (Bendelac et al., 2007;Brennan et al., 2013;Brigl and Brenner, 2004;Kronenberg, 2005;Kumar et al., 2017;Lantz and Bendelac, 1994;Taniguchi et al., 2003). Unlike conventional ab TCRs that recognize peptide antigens presented on classical polymorphic major histocompatibility complex (MHC) Class I and II molecules, the iNKT TCR recognizes glycolipid antigens presented on non-polymorphic MHC Class I-like molecule CD1d (Cohen et al., 2009). iNKT cells in mice comprise CD4 + and CD4 À CD8 À (double negative, DN) subsets (Brigl and Brenner, 2004), and iNKT cells in humans comprise CD4 + , CD8 + , and DN subsets (Brigl and Brenner, 2004). iNKT cells express high levels of cytokine mRNA and produce large amounts of cytokines on primary stimulation (Brigl and Brenner, However, human periphery blood contains extremely low number and high variability of iNKT cells ($0.001-1% in blood), making it challenging to expand sufficient numbers of iNKT cells for therapeutic applications (Krijgsman et al., 2018). To overcome this critical limitation, we have previously established a method to generate large amounts of human iNKT cells through TCR gene engineering of hematopoietic stem cells (HSCs) followed by in vivo reconstitution; using this method, we have successfully generated both mouse and human HSC-engineered iNKT (HSC-iNKT) cells Smith et al., 2015;Zhu et al., 2019). Although such an in vivo approach to providing iNKT cells may be suitable for autologous transplantation, applying this for allogeneic transplantation faces significant hurdles (Smith et al., 2015;Zhu et al., 2019). Here, we intended to build on the HSC-iNKT engineering approach and develop an ex vivo culture method to produce large amounts of third party human iNKT cells; these cells can potentially be used as a ''universal'' and ''off-the-shelf'' reagent for improving allo-HSCT outcomes by ameliorating GvHD while preserving GvL effect.
This manufacturing process of generating HSC-iNKT cells was robust and of high yield and high purity for all 9 donors tested (4 for ATO culture and 5 for feeder-free culture) ( Figure 1D). Based on the results, it was iScience Article estimated that from one quality CB donor (comprising about 1-5 x 10 6 HSCs), about 10 11-10 12 HSC-iNKT cells could be generated that can potentially be formulated into about 10,000-100,000 doses, assuming about 10 7 HSC-iNKT cells per dose ( Figure 1D). The dosage (about 10 7 HSC-iNKT cells per dose) was estimated based on an earlier clinical study, wherein 0.031 3 10 6 CD4 À iNKT cells/kg of body weight was associated with amelioration of GvHD (Chaidos et al., 2012).
To increase the safety profile of the HSC-iNKT cell product, we included an sr39TK PET imaging/suicide gene in the lentiviral vector, which allows for the in vivo monitoring of these cells using PET imaging and the elimination of these cells through ganciclovir (GCV)-induced depletion in case of an adverse event (Figures S1A and S1B). In cell culture, GCV treatment induced effective killing of HSC-iNKT cells (Figures S1B and S1C). In an NSG mouse xenograft model, GCV treatment induced efficient depletion of HSC-iNKT cells from all tissues examined (i.e., blood, liver, spleen and lung) (Figures S1D-S1F). Therefore, the engineered HSC-iNKT cell product is equipped with a potent ''kill switch'', significantly enhancing its safety profile.

Third party HSC-iNKT ( 3rd HSC-iNKT) cells ameliorate Xeno-GvHD in NSG mice engrafted with human PBMC
The engineered HSC-iNKT cells were predominantly CD4 À ( Figures 1B, 1C, and 1E); this subset of human iNKT cells were reported to be associated with reduced GvHD in patients (Chaidos et al., 2012). To test the anti-GvHD potential of 3rd HSC-iNKT cells, we utilized a xeno-GvHD model wherein NSG mice were engrafted with human PBMCs (Shultz et al., 2007). NSG mice were preconditioned with non-lethal total body irradiation (TBI, 100 cGy), and were injected intravenously (i.v.) with healthy donor PBMCs with or without the addition of 3rd HSC-iNKT cells. The recipients were monitored daily for clinical signs of GvHD ( Figure 2A). The addition of 3rd HSC-iNKT cells significantly delayed GvHD onset, reduced body weight loss and prolonged survival ( Figures 2B-2D and 2F). The delayed onset and reduced GvHD severity were associated with the delay of donor T cell expansion in the peripheral blood of experimental mice ( Figure 2E).
To further characterize the changes in GvHD severity, the acute and chronic GvHD overlapping target organs (i.e., lungs, liver, and skin) and chronic GvHD prototypical target organs (i.e., salivary glands) were collected for pathological analysis on day 40 after engrafting donor PBMCs alone or together with 3rd HSC-iNKT cells (Wu et al., 2013). Compared with control NSG mice, the recipient mice engrafted with PBMCs alone showed severe infiltration and damages in the liver and lung. Although the skin tissue did not have severe infiltration, there was a thickened epidermis, a sign of excessive collagen deposition (Wu et al., 2013). The salivary gland also showed infiltration and damage of gland follicles ( Figures 2G-2J). These results suggested that by day 40 after PBMC engraftment, the recipient mice had overlapping iScience Article acute and chronic GvHD. On the other hand, the mice receiving additional 3rd HSC-iNKT cells showed marked reduction in T cell infiltration in the liver, lungs, and salivary glands as well as tissue damage scores ( Figures 2G-2J). Addition of 3rd HSC-iNKT cells also markedly reduced hair loss and epidermal thickening, although T cell infiltration in the skin tissues was mild and no significant difference was observed between recipient mice with or without the addition of 3rd HSC-iNKT cells ( Figures 2G-2J).
Flow cytometry analysis also revealed significantly less numbers of donor T cells in the blood and spleen, as well as less T cell infiltration in GvHD target organs (i.e., lungs, liver and bone marrow; Figures S2A and S2B). Furthermore, intracellular cytokine staining showed that by day 40 after PBMC injection, the addition of 3rd HSC-iNKT cells significantly reduced the proportion of donor CD4 + T cells actively producing Th1-type pro-inflammatory cytokines (i.e., IFN-g and GM-CSF); the proportion of CD4 + T cells producing the Th2type anti-inflammatory cytokine (i.e., IL-4) was not changed ( Figures S2C and S2D). Together, these results suggest that 3rd HSC-iNKT cells suppress the expansion of Th1-type pathogenic donor T cells in target tissues and thereby ameliorating acute and chronic GvHD.

3rd HSC-iNKT cells eliminate donor CD14 + myeloid cells in part through CD1d recognition
Donor myeloid cell-derived antigen presenting cells have been reported to exacerbate acute and chronic GvHD induced by donor T cells (Anderson et al., 2005;Chakraverty and Sykes, 2007;Jardine et al., 2020). Donor T cell production of GM-CSF has also been reported to recruit donor myeloid cells, which in turn amplifies the activation of allogeneic T cells and worsens GvHD severity (Piper et al., 2020;Tugues et al., 2018). Consistently, we observed that removal of CD14 + myeloid cells in the PBMCs reduced xeno-GvHD in NSG recipient mice ( Figures 3A-3H). In contrast, co-injection of donor PBMCs together with 3rd HSC-iNKT cells resulted in a dramatic reduction of donor CD14 + myeloid cells in recipient mice within three days of injection, in tissues spanning blood, lymphoid tissues (i.e, spleen and lymph node), and GvHD target tissues (i.e., liver and lung) ( Figures 3A-3C). Meanwhile, donor T and B cell, which expressed lower levels of CD1d compared to CD14 + myeloid cells, showed no detectable changes ( Figures S4A-S4E).
To study the molecular regulation of 3rd HSC-iNKT cell depletion of donor CD14 + myeloid cells, we performed an in vitro mixed lymphocyte reaction (MLR) assay ( Figure 4A). Healthy donor PBMCs (non-irradiated; as responder representing donor cells) were mixed with donor-mismatched PBMCs (irradiated; as stimulator representing recipient cells) to study alloreaction , with or without the addition of 3rd HSC-iNKT cells. A pair of HLA-A2 positive and negative PBMCs were used to distinguish responders from stimulators ( Figure 4A). In agreement with the in vivo results, in the MLR assay 3rd HSC-iNKT cells effectively ameliorated alloreaction as evidenced by the reduction of IFN-g production ( Figure 4B). Responder PBMCs contained CD14 + myeloid cells expressing high levels of CD1d molecule that can be recognized by iNKT TCR (Bae et al., 2019;King et al., 2018;Li et al., 2022b), corresponding to their efficient depletion by 3rd HSC-iNKT cells ( Figures 4C-4E, S4F, and S4G). On the other hand, human T and B cells from responder PBMCs expressed low levels of CD1d and were not altered by the addition of 3rd HSC-iNKT cells iScience Article ( Figures 4C-4E). Depletion of CD14 + myeloid cells population was significantly alleviated by the addition of anti-CD1d blocking antibody ( Figures 4D and 4E, S4F, and S4G). Taken together, 3rd HSC-iNKT cells ameliorate GvHD through eliminating donor CD14 + myeloid cells at least partly through CD1d recognition.

3rd HSC-iNKT cells preserved GvL activity while ameliorating GvHD
Next, we studied the potential of 3rd HSC-iNKT cells to preserve graft-versus-leukemia (GvL) while ameliorate GvHD, using a human Raji B cell lymphoma and a human HL60 acute myeloid leukemia (AML) xenograft NSG mouse models. We engineered Raji and HL60 tumor cells to overexpress the firefly luciferase and EGFP dual-reporters (denoted as Raji-FG and HL60-FG, respectively) to enable the convenient measurement of tumor killing using in vitro luminescence reading or in vivo bioluminescence imaging (BLI). When co-cultured in vitro, 3rd HSC-iNKT cells effectively killed the Raji-FG and HL60-FG cells via a NK activating receptor (i.e., NKG2D and DNAM-1)-mediated tumor targeting mechanism ( Figures S5A-S5F).
NSG mice were inoculated intravenously (i.v.) with Raji-FG cells, followed by adoptive transfer of healthy donor PBMCs without or with the addition of 3rd HSC-iNKT cells ( Figure 5A). Control NSG mice receiving Raji-FG cells alone died as a result of high tumor burden by day 27 (Figures 5B-5F). Tumor-bearing NSG mice receiving PBMCs with or without the addition of 3rd HSC-iNKT cells showed rapid clearance of the Raji-FG cells (Figures 5B and 5C). However, the tumor-eradicated NSG mice receiving PBMCs all died by day 58 with high clinical GvHD scores, rapid weight loss, and rapid expansion of donor T cells ( Figures 5D-5G). The mice receiving PBMCs together with 3rd HSC-iNKT cells survived significantly longer, for up to 106 days with a much slower progression of GvHD and decline in weight ( Figures 5D-5G). Similar results were obtained from the human HL60 AML xenograft NSG mouse model ( Figures 6A-6G). Taken

. 3rd HSC-iNKT cells ameliorate GvHD through eliminating donor CD14 + myeloid cells through CD1d recognition
In vitro mixed lymphocyte reaction (MLR) assay was performed using healthy donor PBMCs (responders) co-cultured with irradiated donor-mismatched allogeneic PBMCs (stimulators) with or without the addition of 3rd HSC-iNKT cells. Where applicable, purified anti-human CD1d antibody or its IgG isotype control was also added. To identify responders and stimulators by flow cytometry, HLA-A2 + responders and HLA-A2stimulators were used in the study. iScience Article together, these results strongly support the potential of 3rd HSC-iNKT cells to ameliorate GvHD while preserving GvL effect in the treatment of blood cancers.

DISCUSSION
iNKT cells are uniquely positioned at the crossroads of innate and adaptive immunity and have potent immunoregulatory functions in a variety of diseases (Brennan et al., 2013;Van Kaer et al., 2011). Research into harnessing iNKT cells to combat GvHD began decades ago (Lan et al., 2001), but the clinical application of iNKT cells has been hindered by their scarcity in peripheral blood (Krijgsman et al., 2018). We have recently developed an ex vivo HSC-iNKT culture method that can robustly generate large quantities of pure, clonal human iNKT cells (Figure 1 and S1) . The resulting third-party HSC-iNKT ( 3rd HSC-iNKT) cells closely resembled peripheral blood-derived endogenous CD4 À iNKT cells and displayed anti-GvHD activity while preserving GvL effects in preclinical models of leukemia and lymphoma (Figures 2, 3, 4, 5, 6, and S2-S5). Importantly, such 3rd HSC-iNKT cells do not cause GvHD themselves and are resistant to allorejection because of their intrinsic low expression of HLA-I and II molecules , highlighting their potential for off-the-shelf anti-GvHD therapy.
GvHD prophylaxis is centered around calcineurin inhibitor (CNI)-based therapy and investigations into new methods including depleting T cells, modulating T cell co-stimulatory pathways (e.g., checkpoints), enhancing regulatory T cells, targeting T cell trafficking, and altering cytokine pathways iScience Article (Gooptu and Antin, 2021). Despite prophylactic interventions, acute GvHD is a common complication of allo-HSCT, occurring in 30-50% of patients, 14-36% of whom develop severe acute GvHD, and is a major cause of morbidity and mortality (Malard et al., 2020). The current first-line treatment for acute GvHD is systemic steroid therapy, but almost half of all patients will become refractory to treatment and there is no accepted standard-of-care treatment for steroid refractory-acute GvHD (Malard et al., 2020). The dismal survival rate and poor quality of life in these patients highlight the urgent need for novel therapeutic and prophylactic agents against acute GvHD.
The driver of clinical acute GvHD is donor alloreactive T cells (Ball et al., 2008). Following lymphodepletion and HSCT, host and donor antigen-presenting cells respond to host tissue damage and lead to the activation of donor T cells (Ramachandran et al., 2019). Although culpable for GvHD, HSCT-derived T cells are essential for antitumor effects, as their depletion from HSCT grafts precipitates increased relapse rates (Horowitz et al., 1990). To study the anti-GvHD potential of 3rd HSC-iNKT cells, we adopted an xeno-GvHD NSG mouse model, in which human PBMCs are intravenously infused and subsequent donor T cell activation results in GvHD (Figures 2 and S2), replicating some of the components of clinical GvHD (Ali et al., 2012;King et al., 2009).
Although the mechanisms are currently under investigation, our ex vivo culture of iNKT TCR transduced HSCs produces nearly all CD4 À HSC-iNKT cells ( Figures 1B, 1C and 1E) . Like PBMC-derived iScience Article endogenous CD4 À iNKT cells, the engineered HSC-iNKT cells express large amounts of IFN-g and TNF-a as well as Granzyme B and Perforin (Figure 1) , indicative of a Th1 cytokine profile and cytotoxic potential (Li et al., 2021a. In addition, these HSC-iNKT cells display low response to IL-12/IL-18 innate signaling in vitro (Data not shown). In 2012, Chaidos et al. conducted a comprehensive analysis of all immune populations in allogeneic HSCT grafts, and found that only CD4 À iNKT cells were correlated with reduced acute GvHD occurrence (Chaidos et al., 2012). Five years later, Rubio et al. also revealed that only pre-transplant donor CD4 À iNKT cells predicted clinical acute GvHD following HSCT (Rubio et al., 2017). Corroborating the clinical findings, a preclinical study from the same research team confirmed CD4 À iNKT cells, but not CD4 + iNKT cells, prevented GvHD using a xenograft NSG mouse model (Coman et al., 2018), and in vitro assays revealed that CD4 À iNKT cells reduced the maturation and induced the apoptosis of human DCs (Coman et al., 2018). In our study, 3rd HSC-iNKT cells ameliorated GvHD though depleting donor CD14 + myeloid cells, at least partly via CD1d recognition (Figures 3 and 4). Interestingly, CD4 + subpopulation of iNKT cells has also been implicated in GvHD amelioration, albeit through different mechanisms (Chaidos et al., 2012;Coman et al., 2018;Mavers et al., 2017;Rubio et al., 2012). The beneficial role in GvHD has been attributed to IL-4-induced Treg expansion in preclinical syngeneic mouse models (Lan et al., 2003;Pillai et al., 2007;Schneidawind et al., 2014Schneidawind et al., , 2015. One interesting future direction would be modifying our ex vivo HSC-iNKT cell culture to produce CD4 + human iNKT cells to harness the anti-GvHD potential of this subpopulation of iNKT cells. iNKT cells can also play a direct role in tumor killing. Through CD1d dependent and independent means, iNKT cells have been shown to lyse a variety of tumor cells (King et al., 2018;Li et al., 2021b;Zhu et al., 2019). Furthermore, in hematological and solid tumor models, adoptive transfer of iNKT cells reduces tumor burden and enhances overall survival (Fujii et al., 2013). Our previous studies have demonstrated the antitumor functions of HSC-iNKT cells in vivo when targeting CD1d positive and negative cancer cells Zhou et al., 2021). Importantly, HSC-iNKT cells do not recognize mismatched MHCs and thus pose no risk of inducing GvHD; furthermore, because of their intrinsic low expression of HLA-I and II molecules, these cells are resistant to allorejection . These features of HSC-iNKT cells make them suitable for allogeneic cell therapy.
Allo-HSCT is an established, effective treatment for hematological malignancies, but GvHD is a common and debilitating adverse event for many allo-HSCT recipients. We propose to develop the off-the-shelf HSC-iNKT cell therapy to ameliorate GvHD while preserving GvL in the treatment of blood cancers. The reported ex vivo HSC-iNKT cell culture is robust and of high yield and purity, with the potential of being scaled for further translation and clinical development. From one cord blood donor, over 10,000 doses of third-party HSC-iNKT cells can be manufactured and cryopreserved for ready distribution to allo-HSCT patients; MHC matching is not needed. This study highlights the potential of 3rd HSC-iNKT cells to address a critical unmet medical need and warrants further investigations of this promising off-the-shelf cell product.

Limitations of the study
Predominant mouse models studying GvHD typically employ transplantation of T cell-depleted bone marrow and donor-derived T cells into lethally irradiated recipients; these are paramount to advance the forefront of knowledge regarding the incidence of GvHD within allo-HSCT therapeutics (Schroeder and Di-Persio, 2011). In this study, healthy donor T cells were used to generate a PBMC-xenograft NSG mouse model, producing a construct where T cell-mediated GvHD could be studied and manipulated in vivo.
However, limitations to this model preclude its ability to fully reflect GvHD pathology in allo-HSCT. Such complexity arises from factors such as the restricted availability of human embryonic tissue for transplant, the need for sublethal total body irradiation, demand for a high quantity of human PBMCs, and instability in the onset window of GvHD (Huang et al., 2018). In addition, murine immunoreaction after engraftment of human immune cells is highly distinct compared to that in humans in regard to both biological phenotype and genetics. Therefore, developing a model that more accurately mimics human GvHD pathology, while reducing variance from these limitations, is necessary to understand patient reactivity to allo-HSCT therapies.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank the University of California, Los Angeles (UCLA) animal facility for providing animal support; the UCLA Translational Pathology Core Laboratory (TPCL) for providing histology support; the UCLA CFAR Virology Core for providing human cells; and the UCLA BSCRC Flow Cytometry Core Facility for cell sorting support. This work was supported by a Director's New Innovator Award from the NIH (DP2 CA196335, to L.Y.

Cell lines and viral vectors
The murine bone marrow derived stromal cell line MS5-DLL4 was obtained from Dr. Gay Crooks' lab (UCLA). Human Raji B cell lymphoma cell line, HL60 acute myeloid leukemia cell line, and HEK 293 T cell line were purchased from the AmericanType Culture Collection (ATCC).
Lentiviral vectors used in this study were all constructed from a parental lentivector pMNDW Zhu et al., 2019). The Lenti/iNKT-sr39TK vector was constructed by inserting into pMNDW vector a synthetic tricistronic gene encoding human iNKT TCRa-F2A-TCRb-P2A-sr39TK; the Lenti/FG vector was constructed by inserting into pMNDW a synthetic bicistronic gene encoding Fluc-P2A-EGFP. The synthetic gene fragments were obtained from GenScript and IDT. Lentiviruses were produced using HEK 293T cells, following a standard calcium precipitation protocol and an ultracentrifigation concentration protocol Zhu et al., 2019). Lentivector titers were measured by transducing HT29 cells with serial dilutions and performing digital qPCR Zhu et al., 2019).
To make stable tumor cell lines overexpressing firefly luciferase and enhanced green fluorescence protein (FG) dual-reporters, parental tumor cell lines were transduced with lentiviral vectors encoding the intended gene(s). 72h following lentiviral transduction, cells were subjected to flow cytometry sorting to isolate gene-engineered cells for making stable cell lines. Two stable tumor cell lines were generated for this study, including Raji-FG and HL60-FG.

Human periphery blood mononuclear cells (PBMCs)
Healthy donor human PBMCs were obtained from the UCLA/CFAR Virology Core Laboratory, with identification information removed under federal and state regulations. Cells were cryopreserved in Cryostor CS10 (BioLife Solutions) using CoolCell (BioCision) and were stored in liquid nitrogen for all experiments and long-term storage.

METHOD DETAILS
Antibodies and flow cytometry